Anti mouse human ki 67 11f6

Frozen OCT blocks of tumors and LNs were cut using a cryostat (Leica) into 8‐μm thick sections and cells were incubated in glass‐bottomed well plate (Lab Tek™). Meanwhile, cell samples were also stained by the following procedure. The samples were stained using anti‐pan‐cytokeratin (AE1/AE3, sc‐81,714, SCBT), anti‐Melan‐A (ab210546, Abcam), anti‐LYVE‐1 (ab14917, Abcam), anti‐fibronectin (NBP1‐91258, Novus Biologicals), anti‐CD31 (14–0311‐82, Invitrogen), anti‐caspase‐3 (4‐1‐18, BioLegend), anti‐mouse/human Ki‐67 (11F6, BioLegend) antibodies, or anti‐HSP70 (HPA052504, Sigma‐Aldrich). Dye‐conjugated secondary antibodies (711‐585‐152, 711‐545‐152, 712‐545‐150, 712‐585‐153, Jackson ImmunoResearch) were used for the addition of the fluorescence signals. DAPI (VECTASHIELD, Vector Laboratories Burlingame, CA) was used to stain the cell nuclei. The stained tissue sections were imaged using a fluorescent confocal microscope and an EVOS FL2 auto microscope or FluoView FV‐10i Olympus Laser Point Scanning Confocal Microscope (Olympus, Center Valley, PA). After splitting each color channel, the fluorescent area for each color channel was measured with ImageJ (National Institutes of Health; Bethesda, MD). The expression level for each marker was calculated on the basis of normalization with the DAPI signal.